The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

Bottom Line:
Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

ABSTRACTWe have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

Figure 4: Live cell imaging of spc24-1 cells containing GFP-Spc42p, an SPB component, and GFP-CEN5. CEN5 was labeled by integration of tet operators (which bind GFP-tet repressors) 1.4 kb from CEN5 (Tanaka et al. 2000). Cells were synchronized in G1 with α-factor and released at 23°C for 1.5 h (a) or at 36°C (b1–b3, c1–c2) for the times indicated; arrowheads show the SPBs which are brighter than the centromeres (He et al. 2000; Tanaka et al. 2000). The same cell is imaged in b1–b3 and another cell in c1–c2. Bar, 2 μm.

Mentions:
We also examined the behavior of GFP-labeled centromeres and SPBs (He et al. 2000; Tanaka et al. 2000) in live synchronized spc24-1 cells. At 23°C and 1.5 h after release from the α-factor block centromeres had split (Fig. 4 a). They then oscillated normally back and forth between the two Spc42p spots (He et al. 2000; Tanaka et al. 2000) before finally dissociating to the poles during anaphase. At 36°C, however, centromeres did not split during mitosis (Fig. 4 b, 1–3 and c 1–2), as was also observed in ndc10-1 (Tanaka et al. 2000). There were relative movements of the unsplit centromeres and the poles (Fig. 4b 1–2), but it was difficult to tell whether this was microtubule based or whether it reflected the constant movement of the spindle, possibly mediated in part by the cytoplasmic microtubules relative to a stationary unconnected centromere. There also appeared to be lateral displacements of the centromeres from the spindle axis, suggesting a detachment of centromeres from the spindle. This was quantified in cells with all three dots in focus, which would actually bias against detecting lateral displacement. At 1.25–1.75 h the average lateral displacement (measured as distance r in He et al. 2000) was 0.3 ± 0.2 μm (n = 67), which is within the range for wild-type cells (He et al. 2000). However, at 2–2.75 h the lateral displacement was 0.6 ± 0.35 μm (n = 63). These cells were examined by immunofluorescence with antitubulin and anti-GFP to check whether microtubules were connected to individual centromeres that showed a large lateral displacement. For antitubulin we used the mAb YOL1/34 which can detect individual microtubules by immunofluorescence in 3T3 cells (Kilmartin et al. 1982); no connecting microtubules were detected (data not shown). The nuclear microtubules remained in the tight bundles seen by immunofluorescence. These results might suggest detachment of the centromeres from the spindle in spc24-1 cells. However, the detachment is only seen later in the block and thus may be indirect, and also microtubules connecting such centromeres to the pole could be particularly unstable during processing for immunofluorescence and difficult to detect.

Figure 4: Live cell imaging of spc24-1 cells containing GFP-Spc42p, an SPB component, and GFP-CEN5. CEN5 was labeled by integration of tet operators (which bind GFP-tet repressors) 1.4 kb from CEN5 (Tanaka et al. 2000). Cells were synchronized in G1 with α-factor and released at 23°C for 1.5 h (a) or at 36°C (b1–b3, c1–c2) for the times indicated; arrowheads show the SPBs which are brighter than the centromeres (He et al. 2000; Tanaka et al. 2000). The same cell is imaged in b1–b3 and another cell in c1–c2. Bar, 2 μm.

Mentions:
We also examined the behavior of GFP-labeled centromeres and SPBs (He et al. 2000; Tanaka et al. 2000) in live synchronized spc24-1 cells. At 23°C and 1.5 h after release from the α-factor block centromeres had split (Fig. 4 a). They then oscillated normally back and forth between the two Spc42p spots (He et al. 2000; Tanaka et al. 2000) before finally dissociating to the poles during anaphase. At 36°C, however, centromeres did not split during mitosis (Fig. 4 b, 1–3 and c 1–2), as was also observed in ndc10-1 (Tanaka et al. 2000). There were relative movements of the unsplit centromeres and the poles (Fig. 4b 1–2), but it was difficult to tell whether this was microtubule based or whether it reflected the constant movement of the spindle, possibly mediated in part by the cytoplasmic microtubules relative to a stationary unconnected centromere. There also appeared to be lateral displacements of the centromeres from the spindle axis, suggesting a detachment of centromeres from the spindle. This was quantified in cells with all three dots in focus, which would actually bias against detecting lateral displacement. At 1.25–1.75 h the average lateral displacement (measured as distance r in He et al. 2000) was 0.3 ± 0.2 μm (n = 67), which is within the range for wild-type cells (He et al. 2000). However, at 2–2.75 h the lateral displacement was 0.6 ± 0.35 μm (n = 63). These cells were examined by immunofluorescence with antitubulin and anti-GFP to check whether microtubules were connected to individual centromeres that showed a large lateral displacement. For antitubulin we used the mAb YOL1/34 which can detect individual microtubules by immunofluorescence in 3T3 cells (Kilmartin et al. 1982); no connecting microtubules were detected (data not shown). The nuclear microtubules remained in the tight bundles seen by immunofluorescence. These results might suggest detachment of the centromeres from the spindle in spc24-1 cells. However, the detachment is only seen later in the block and thus may be indirect, and also microtubules connecting such centromeres to the pole could be particularly unstable during processing for immunofluorescence and difficult to detect.

Bottom Line:
Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

ABSTRACTWe have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.